Determination of the three-dimensional structures of RNA molecules, RNA-protein and DNA-protein complexes with high resolution is invaluable for gaining understanding of biological systems at the molecular level. (See for example, refs. 1-4). X-ray crystallography is the most direct and powerful tool for structure determination of these macromolecules. (Refs. 5-7). However, derivatization with heavy atoms for phase determination, a long-standing problem in nucleic acid X-ray crystallography, has impeded the structural determination process. (Refs. 8, 9). It can take years just to prepare derivatives and to determine the required phase information using traditional approaches.
Currently, derivatization approaches include heavy-atom soaking of crystals, co-crystallization, and halogen-derivatization of oligonucleotides. Heavy-atom soaking and co-crystallization have proven to be much more difficult for nucleic acids than for proteins, probably because nucleic acids lack specific metal ion binding sites. 5-halogen-uridine (5-bromine or iodine) or 5-halogen-deoxyuridine (thymidine mimic) is used to derivative nucleic acids for phase determination. As these halogenated nucleotides are not very stable under X-ray or UV irradiation, long exposure may cause decomposition. (Ref. 10). In the case of iodine derivatives, isomorphism is a requirement for the Multiple Isomorphous Replacement (MIR) technique, but crystal structures of iodine derivatives not always isomorphous with (i.e. do not adopt the same molecular conformation as) the native structures, (see for example, Refs. 9 and 11) which limits the usefulness of the iodine derivatives in structural determination.
The Multiwavelength Anomalous Dispersion (MAD) technique has been developed for structure determination of macromolecules using synchrotron and anomalous scattering atoms. (Refs. 12 and 14). The synchrotron radiation provides the required X-ray wavelengths, and anomalous scatterers, such as selenium or bromine, can provide distinctive diffraction pattern for phase determination. As phasing signal of bromine is relatively weaker than that of selenium, more bromine atoms need to be incorporated into large nucleic acid molecules in order to successfully use MAD phasing. Incorporation of many bromine atoms, with limited choice of positioning, can cause significant changes in native structures. (Refs. 9 and 11). Bromine derivatives, used in current MAD phasing, are thereby more limited to structure determination of small oligonucleotides.
Another problem with bromine derivatives is the limited choice of positioning, and even where substitution is possible, structural perturbation is difficult to avoid. (Refs. 9, 11). Therefore, there is a need for alternative derivatives that require incorporation of a few heavy atoms, and allow choice of heavy atom positioning to avoid structural perturbation, which is especially important for labeling large nucleic acid molecules for MAD phasing.
Nucleic acids (Designated as structure 1 in FIG. 1) may be prepared by solid-phase synthesis using phosphoramidites (Structure 2), in vitro RNA transcription or DNA polymerization using triphosphates (Structure 3) as building blocks according to well known methods. See for example, Gait, M. J. (1991) DNA/RNA synthesis and labeling, Curr. Opin. Biotechnol. 2(1): 61-68; and Sproat, B. S. (1995) Chemistry and applications of oligonucleotide analogues, J. Biotechnol. 41 (2-3): 221-238.
There is a need for methods whereby any one of the oxygen atoms of a nucleotide unit, including 2′, 3′, 5′, and α-phosphate oxygen atoms, the ring oxygen atom, and oxygen atoms of the nucleobases, may be selectively replaced by selenium. Such methods would be particularly valuable in offering a choice for positioning selenium atoms, especially if this could be achieved without structural perturbation in nucleic acids. These molecules would then be useful for determinations of the native structures without the Selenium modification.
Selenium is an essential trace element for humans. Statistic data and survey indicate that people die from lack of selenium in some parts of the world. Though a limited amount of research has addressed the metabolism of selenium in humans, much is known about how experimental animals regulate selenium. It is reported that selenium deficiency increases the pathology of an influenza virus infection. Beck, M. A.; Nelson, H. K.; Shi, Q.; Van Dael P.; Schiffrin, E. J.; Blum, S.; Barclay, D.; Levander, O. A., “Selenium deficiency increases the pathology of an influenza virus infection”, J. FASEB 2001, 15, 1481-1483.
In a mouse model, it was also observed that a benign strain of coxsackievirus B3 became virulent and caused myocarditis in selenium- and vitamin E-deficient mice. Beck, M. A.; Levander, O. A., “Host nutritional status and its effect on a viral pathogen”, J. Infect Dis. 2000, 182 Suppl 1:S93-96. This change in pathogenicity was due to mutations in the viral genome, which changed an avirulent virus into a virulent one. Once these mutations occurred, even mice with normal nutrition developed disease from the mutated virus.
These results suggest that the oxidative stress status and selenium level of the host can have a profound influence on a viral pathogen. Pathogenesis of mycobacterial disease in HIV-infected people is also influenced by selenium status. Shor-Posner, G.; Miguez, M. J.; Pineda, L. M.; Rodriguez, A.; Ruiz, P.; Castillo, G.; Burbano, X.; Lecusay, R.; Baum, M., “Impact of selenium status on the pathogenesis of mycobacterial disease in HIV-1-infected drug users during the era of highly active antiretroviral therapy”, J Acquir Immune Defic Syndr 2002, 29, 169-173.
Selenium supplementation has been reported to suppress carcinogenesis in many animal models. Finley, J. W; Ip, C.; Lisk, D. J.; Davis, C. D.; Hintze, K, J.; Whanger, P. D., “Cancer-Protective Properties of High-Selenium Broccoli”, J. Agric. Food Chem. 2001, 49, 2679-2683. The cancer protective effect of dietary selenium in humans is also supported by intervention trials as well as by epidemiological data. Manar, M. J., MacPherson, G. D.; Mcardle, F.; Jackson, M. J.. Hart, C. A., “Selenium status, kwashiorkor and congestive heart failure”, Acta Paediatr 2001, 90, 950-952.
Hence, there is a need for selenium nucleoside, selenium nucleotides and selenium derivatives of nucleic acids as food supplements. Such selenium derivatives would be valuable as anticancer agents.